*NCBI Gene # or RefSeq#: 3656220 (Gene #) *Protein ID (NP or XP #) or Wolbachia#: 15644 *Organism (including strain): Trypanosoma brucei Etiologic Risk Group (see link below): Parasitic Agents *Background/Disease Information (sort of like the Intro to your Mini Research Write up): Trypanosoma brucei is a protozoan that causes African trypanosomiasis (sleeping sickness). This disease can be found in humans or animals. It is transmitted by the tsetse fly. African trypanosomiasis can be treated with medication. Essentiality of this protein: http://www.jbc.org/content/279/5/3420.long
This protein was found to be essential in inhibiting T. brucei during an experiment done with mice (see article). Complex of proteins?: Druggable Target: No
-- List cost and quantity of substrate reagents and supplier
$237.00 from Sigma Aldrich Structure Available (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: 3EMJ -- For Homology Model option: ---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
Query 74 RRTRAKLELVKEEPHN--PIAQDTLKKEGNAL---RFFKYG-DVPFNYGFAPQTWEDPSV 127 + ++ ++ E P N PI + KE AL RF + P NYGF P T Sbjct 10 KANNNEINVIIEIPMNSGPIKYE-FDKESGALFVDRFMQTTMSYPCNYGFIPDTL----- 63
Query 128 MDQLTTCGGDGDPIDIVELSSNPFAVGSVRAVRVLGLLGLIDEGETDWKVITEAIGP-DA 186 DGDP+D++ ++ +P GSV R +G+L + DE D K+I D Sbjct 64 -------SNDGDPVDVLVVAHHPVVPGSVIKCRAIGVLMMEDESGLDEKIIAVPTSKLDI 116
Query 187 TGTY-GSLNNVPQELKATIVKWFREYK 212 T + L+++ + LK IV +F YK Sbjct 117 TFDHIKELDDLCEMLKKRIVHFFEHYK 143
---- Query Coverage: 56% ---- Max % Identities: 56% ---- % Positives: 55% ---- Chain used for homology: 3EMJ
Current Inhibitors: about 81 current inhibitors Expression Information (has it been expressed in bacterial cells): Yes it can be expressed in E.coli Purification Method: Recombinant protein using His-TAG Image of protein (PyMol with features delineated and shown separately): (Image of inorganic phosphatase for Rickettsia prowazekii due to unavailability of protein in Trypanosoma brucei) *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): mrptsimfkgmtgagimlpawalqevgaagtrawrmyftsseagsvarrsawhdlplhps pdasvitfvceiprrtraklelvkeephnpiaqdtlkkegnalrffkygdvpfnygfapq twedpsvmdqlttcggdgdpidivelssnpfavgsvravrvlgllglidegetdwkvite aigpdatgtygslnnvpqelkativkwfreyktadgkkpnefvfggelrnaddalrvieg gsrqytgliagtvrnpgywlh *length of your protein in Amino Acids: 261 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 28675.5 Molar Extinction coefficient of your protein at 280 nm wavelength: 49055 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. *CDS Gene Sequence (paste as text only): >gi|72386904|ref|XM_838784.1| Trypanosoma brucei inorganic pyrophosphatase, putative (Tb927.3.2840) partial mRNA ATGCGCCCAACATCAATTATGTTTAAAGGGATGACCGGAGCAGGCATTATGCTCCCCGCATGGGCATTGC AGGAAGTGGGAGCAGCGGGAACGCGGGCGTGGCGCATGTACTTCACAAGCAGCGAGGCCGGATCCGTTGC GAGGCGCTCGGCATGGCATGATCTTCCGCTACATCCATCACCTGATGCATCTGTCATTACATTTGTATGT GAGATTCCCAGAAGGACGCGTGCCAAATTGGAACTCGTGAAGGAAGAACCGCACAACCCGATAGCTCAAG ACACATTAAAGAAAGAGGGCAATGCTTTGCGGTTCTTCAAGTATGGTGATGTCCCCTTCAACTATGGTTT TGCGCCACAAACATGGGAGGATCCTTCAGTAATGGACCAACTGACGACGTGTGGGGGTGATGGTGATCCC ATTGATATCGTTGAATTATCGTCAAATCCCTTTGCAGTTGGTTCCGTAAGGGCAGTTCGAGTCCTCGGGT TGTTGGGACTTATTGACGAGGGGGAAACCGACTGGAAAGTCATTACGGAGGCCATTGGACCCGATGCGAC CGGTACGTACGGGTCTTTAAATAATGTGCCACAAGAGCTGAAGGCTACCATTGTTAAATGGTTCCGCGAG TACAAAACTGCTGATGGGAAGAAGCCAAATGAATTTGTTTTTGGAGGGGAGTTGCGTAATGCAGACGACG CACTTCGTGTGATTGAAGGAGGTTCCCGCCAGTACACCGGACTTATTGCAGGCACGGTTCGTAATCCCGG TTACTGGCTTCACTGA *GC% Content for gene: *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
*Target (protein/gene name): Inorganic pyrophosphatase
*NCBI Gene # or RefSeq#: 3656220 (Gene #)*Protein ID (NP or XP #) or Wolbachia#: 15644
*Organism (including strain): Trypanosoma brucei
Etiologic Risk Group (see link below): Parasitic Agents
*Background/Disease Information (sort of like the Intro to your Mini Research Write up): Trypanosoma brucei is a protozoan that causes African trypanosomiasis (sleeping sickness). This disease can be found in humans or animals. It is transmitted by the tsetse fly. African trypanosomiasis can be treated with medication.
Essentiality of this protein: http://www.jbc.org/content/279/5/3420.long
This protein was found to be essential in inhibiting T. brucei during an experiment done with mice (see article).
Complex of proteins?:
Druggable Target: No
*EC#: 3.6.1.1
Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.6.1.1
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/ionorgpyrophosph.pdf
-- link (or citation) to paper that contains assay information
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/ionorgpyrophosph.pdf
-- List cost and quantity of substrate reagents and supplier
$237.00 from Sigma Aldrich
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 3EMJ
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
Query 74 RRTRAKLELVKEEPHN--PIAQDTLKKEGNAL---RFFKYG-DVPFNYGFAPQTWEDPSV 127
+ ++ ++ E P N PI + KE AL RF + P NYGF P T
Sbjct 10 KANNNEINVIIEIPMNSGPIKYE-FDKESGALFVDRFMQTTMSYPCNYGFIPDTL----- 63
Query 128 MDQLTTCGGDGDPIDIVELSSNPFAVGSVRAVRVLGLLGLIDEGETDWKVITEAIGP-DA 186
DGDP+D++ ++ +P GSV R +G+L + DE D K+I D
Sbjct 64 -------SNDGDPVDVLVVAHHPVVPGSVIKCRAIGVLMMEDESGLDEKIIAVPTSKLDI 116
Query 187 TGTY-GSLNNVPQELKATIVKWFREYK 212
T + L+++ + LK IV +F YK
Sbjct 117 TFDHIKELDDLCEMLKKRIVHFFEHYK 143
---- Query Coverage: 56%
---- Max % Identities: 56%
---- % Positives: 55%
---- Chain used for homology: 3EMJ
Current Inhibitors: about 81 current inhibitors
Expression Information (has it been expressed in bacterial cells): Yes it can be expressed in E.coli
Purification Method: Recombinant protein using His-TAG
Image of protein (PyMol with features delineated and shown separately): (Image of inorganic phosphatase for Rickettsia prowazekii due to unavailability of protein in Trypanosoma brucei)
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
mrptsimfkgmtgagimlpawalqevgaagtrawrmyftsseagsvarrsawhdlplhps
pdasvitfvceiprrtraklelvkeephnpiaqdtlkkegnalrffkygdvpfnygfapq
twedpsvmdqlttcggdgdpidivelssnpfavgsvravrvlgllglidegetdwkvite
aigpdatgtygslnnvpqelkativkwfreyktadgkkpnefvfggelrnaddalrvieg
gsrqytgliagtvrnpgywlh
*length of your protein in Amino Acids: 261
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 28675.5
Molar Extinction coefficient of your protein at 280 nm wavelength: 49055
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
>gi|72386904|ref|XM_838784.1| Trypanosoma brucei inorganic pyrophosphatase, putative (Tb927.3.2840) partial mRNA
ATGCGCCCAACATCAATTATGTTTAAAGGGATGACCGGAGCAGGCATTATGCTCCCCGCATGGGCATTGC
AGGAAGTGGGAGCAGCGGGAACGCGGGCGTGGCGCATGTACTTCACAAGCAGCGAGGCCGGATCCGTTGC
GAGGCGCTCGGCATGGCATGATCTTCCGCTACATCCATCACCTGATGCATCTGTCATTACATTTGTATGT
GAGATTCCCAGAAGGACGCGTGCCAAATTGGAACTCGTGAAGGAAGAACCGCACAACCCGATAGCTCAAG
ACACATTAAAGAAAGAGGGCAATGCTTTGCGGTTCTTCAAGTATGGTGATGTCCCCTTCAACTATGGTTT
TGCGCCACAAACATGGGAGGATCCTTCAGTAATGGACCAACTGACGACGTGTGGGGGTGATGGTGATCCC
ATTGATATCGTTGAATTATCGTCAAATCCCTTTGCAGTTGGTTCCGTAAGGGCAGTTCGAGTCCTCGGGT
TGTTGGGACTTATTGACGAGGGGGAAACCGACTGGAAAGTCATTACGGAGGCCATTGGACCCGATGCGAC
CGGTACGTACGGGTCTTTAAATAATGTGCCACAAGAGCTGAAGGCTACCATTGTTAAATGGTTCCGCGAG
TACAAAACTGCTGATGGGAAGAAGCCAAATGAATTTGTTTTTGGAGGGGAGTTGCGTAATGCAGACGACG
CACTTCGTGTGATTGAAGGAGGTTCCCGCCAGTACACCGGACTTATTGCAGGCACGGTTCGTAATCCCGG
TTACTGGCTTCACTGA
*GC% Content for gene:
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Resources:
See ProtocolTargetDiscoveryVDS.docx for moreEtiologic Risk Group Categories (for pathogens): http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
Databases of genes/organisms:
http://www.niaid.nih.gov/Pages/default.aspx
http://www.nmpdr.org/FIG/wiki/view.cgi/Main/EssentialGenes
http://tubic.tju.edu.cn/deg/
http://csgid.org/csgid/cake/pages/community_request_gateway
http://tdrtargets.org/
http://gsc.jcvi.org/status.shtml
Gene Information:
NCBI GENE Page: http://www.ncbi.nlm.nih.gov/gene
BLAST Page: http://blast.ncbi.nlm.nih.gov/
Protein Information:
NCBI Protein Page: http://www.ncbi.nlm.nih.gov/protein
Protein Expression Website
Protein Expression Paper: SGC_ProteinProductionPurificationNatMethods2008.pdf